Development Of Fiber Carrier Fixed-bed Bioreactor And Application In Virus Vaccine Production

Fixed-bed bioreactors were widely used for cell cultivation due to distinguishing features of low shear force,high surface area,and low cell inoculation density.However,the disadvantages,such as lack of ideal scale-down platform,complex structure,inhomogeneous cell distribution and foaming aeration limit their applications in biological manufacturing.To solve those above-mentioned problems,a novel scale-down model of fixed-bed bioreactor(tube-fixed-bed bioreactor,TFB bioreactor)and a novel fixed-bed bioreactor(Xcell bioreactor)were developed in this dissertation.In order to obtain homogeneous cell distribution,the structure and operation of the Xcell bioreactor were optimized.Bubble-free aeration of the Xcell bioreactor was achieved by using a novel system with waterfall oxygenation.By optimizing Vero cells culture process and analyzing Vero cells expansion and metabolism in the TFB bioreactor and the Xcell bioreactor with either the serum-containing medium or the serum-free medium,feasibility of Vero cells cultivation process scale-up from the TFB bioreactor to the Xcell bioreactor was confirmed.Based on the TFB bioreactor and the Xcell bioreactor,the fixed-bed bioreactor scale-down/scale-up platform was established.Using this platform,the development and scale-up of Vero cell-based pseudorabies virus(PRV)production process were completed quickly.This platform provides a new strategy for the research and development of PRV vaccine.Finally,we verified that this platform suitable for process development of serum-free PRV production with the Quality by Design(QbD)approach.The results provide a new method and process for the rapid development and scale-up of the production process of cell-based vaccine in China.The major conclusions are as follows:(1)Establishment and application of a scale-down platform of the fixed-bed bioreactors.The tube-fixed-bed bioreactor(TFB bioreactor),consisting of the TubeSpin bioreactor,macrocarriers,and novel stent,was developed as a scale-down platform of fixed-bed bioreactor.The performance of Vero cells in the TFB bioreactor was verified.Compared with microcarrier-based cell cultivation,the TFB biorector yields higher cell density and more suitable for low inoculation density process and serum-free cultivation.In addition,the TFB bioreactor is also suitable for cultivation of CHO cells and 293T cells.The TFB bioreactor worked as a scale-down model of fixed-bed bioreactor for adherent cells cultivation.(2)Design and verification of the novel fixed-bed bioreactors.To solve the problems of complex structure,inhomogeneous cell distribution,and foaming aeration of available fixed-bed bioreactor,two novel laboratory-scale fixed-bed bioreactors,Xcell bioreactor(1-L and 5-L),were developed,based on the data of the TFB bioreactor and criteria of bioreactor scale-up/scale-down.The bubble-free aeration of the Xcell bioreactor was achieved by using novel and simple structure waterfall oxygenation.Its k_La fulfils the requirement of animal cells cultivation,and its mixing time is comparable with that of the STR bioreactor.Thus,the Xcell bioreactor provides good performance of oxygen transfer and mixing.By using hover inoculation technology,the cell capture ability of the Xcell bioreactor is good and cells distributed homogeneously in the fixed-bed bioreactor.With biological experiments,we observed comparable growth curves and glucose consumption rates in the Xcell bioreactor and the TFB bioreactor,which confirms the Vero cells cultivation process can be successfully scaled up from the TFB bioreactor to the Xcell bioreactor.Thus the fixed-bed bioreactor scale-down/scale-up platform was established,which can be used for the optimization and scale-up of cells cultivation process.(3)Application of the novel fixed-bed bioreactor for PRV vaccine production.By using the fixed-bed bioreactor scale-down/scale-up platform,the Vero cell-based PRV production process parameters(TOI(time of infection),MOI(multiplicity of infection)and harvest process)were optimized in the TFB bioreactor.The optimized process was scaled up to the Xcell 1-L bioreactor and the Xcell 5-L bioreactor.Thus the PRV production processes(serum-containing process,serum-free process,serum-containing low inoculation density peocess,serum-free low inoculation density peocess)in fixed-bed bioreactor were established.Compared with commercial PRV vaccine production process with roller bottles,the PRV production process in this study is highly automatic,stable and safe.By using the fixed-bed bioreactor scale-down/scale-up platform,the development of PRV production process was completed efficiently.This study provides a new method for the development and production of PRV vaccine process,and also offered valuable reference for the development of other cell-based vaccines.(4)Process development of serum-free PRV production with the QbD approach.The PRV titer in the harvest medium was consider as Critical Quality Attribute(CQA)and the risk analysis of serum-free PRV vaccine production process in the fixed-bed bioreactor was performed using Ishikawa diagram combined with Failure Mode Effects Analysis(FMEA).The medium,initial inoculation cell density,TOI and MOI,which ranked high with risk priority number(RPN),were considered as Critical Process Parameters(CPPs).By using the TFB bioreactor,the comparative experiment was performed to screen commercial Vero cells in the serum-free media,and the other CPPs were taken into design of experiment(DoE)methodology.The prediction model was established by fitting the DoE data with multiple linear regression.Then the design space and robust setpoints of PRV production process parameters were established.The robust setpoints were confirmed in the TFB bioreactor and then scaled up to the Xcell 1-L bioreactor successfully.Combinatiton of the QbD approach and the fixed-bed bioreactor scale-down/scale-up platform could make the development of stable virus production process fast and efficient.